| Background: Liver fibrosis is a reversible course in many chronic liver diseases but can lead to mortality via development of cirrhosis.Central to the pathogenesis of the disease is hepatic stellate cell(HSC)activation and subsequent conversion into myofibroblasts,which increases the cell capacity for excessive extracellular matrix(ECM)production.However,the relevant mechanismsin remain underexplored,and thus far,there is no effective treatment for liver fibrosis.Enhancer of Zeste Homolog 2(EZH2)has been demonstrated to play a significant role in myofibroblast transdifferentiation;however,the underlying mechanisms remain largely unaddressed.Carnosol(CS),a compound extracted from rosemary,has been shown to have anti-inflammatory,anti-oxidant and anti-cancer properties;however,the effect on the treatment of liver fibrosis has not been reported.Objective: To investigate the anti-fibrotic effect of CS during the progression of liver fibrosis;to explore the signaling mechanisms of Sirtuin 1(SIRT1)/EZH2 in liver fibrosis.Methods:1.60 male Sprague-Dawley rats were kept in standard laboratory conditions,allowed to adapt to the new environment for one week,and then randomly divided into5 groups:(1)control,(2)control+CS(30 mg/kg/d),(3)CCl4,(4)CCl4+CS(15 mg/kg/d),and(5)CCl4+CS(30 mg/kg/d).We generated CCl4-induced liver fibrosis via intraperitoneal injection of CCl4(0.5 mg/kg,diluted 1:10 in olive oil)twice weekly in rats.Meanwhile,CS and olive oil were administered via gavage every day.After 4weeks,all animals were euthanatized,liver and blood samples were harvested for further analysis.Blood samples were collected for measuring Alanine Aminotransferase(ALT),Aspartate Aminotransferase(AST)and the liver tissues were harvested for histopathologic(hematoxylin-eosin,HE and Masson trichrome staining)assessment.The liver protein expressions of SIRT1,EZH2,Collagen I,α-SMA,E-Cadherin and Vimentin were determined by Western Blot.The liver m RNA levels of PPARγ、TIMP1、CTGF、Collagen I and α-SMA were tested by real-time PCR.2.(1)AML-12 and LX-2 cells were divided into four groups: control group,CS(10μM)group,TGFβ1(4ng/ml)group and TGFβ1(4ng/ml)+CS(10μM)group.Cells were pretreated with CS for 6 h before exposure to TGFβ1 for 12 h.The protein levels of Collagen I and α-SMA were examined by Western Blot.(2)AML-12 and LX-2 cells were divided into five groups including control group,TGFβ1(4ng/ml)group and TGFβ1(4ng/ml)+CS(2.5μM,5μM,10μM)groups.Cells were pretreated with 2.5,5,or10 μM CS for 6 h and then exposed to TGFβ1 for 12 h.Then,the levels of Collagen I and α-SMA were determined by Western Blot.(3)Primary hepatic stellate cells(HSCs,isolated from normal livers of 350 g adult male Sprague-Dawley rats)and AML-12 cells were divided into four groups: negative control(si-control)group,si-control+TGFβ1(4ng/ml)group,si-EZH2 group and si-EZH2+CS(10μM)group.HSCs and AML-12 cells were transfected with control si RNA or EZH2 si RNA for 48 h before exposure to TGFβ1for 12 h.The levels of EZH2,PPARγ,TIMP1,CTGF,Collagen I and α-SMA in HSCs were measured with real-time PCR.Collagen I,α-SMA,E-Cadherin and Vimentin protein expressions in AML-12 cells were assayed by Western Blot.Immunofluorescence(×400)analysis of E-Cadherin and Vimentin protein expression was tested in AML-12 cells.(4)HSCs and AML-12 cells were divided into five groups including negative control(si-control)group,si-control+TGFβ1(4ng/ml)group,TGFβ1(4ng/ml)+CS(10μM)group,si-SIRT1+TGFβ1(4ng/ml)group,and si-SIRT1+TGFβ1(4ng/ml)+CS(10μM)group.HSCs and AML-12 cells were transfected with control si RNA or SIRT1 si RNA for 48 h before treatment with CS for 6 h,and the transfected cells were exposed to TGFβ1 for 12 h.The m RNA levels of Collagen I,α-SMA,PPARγ,TIMP1 and CTGF were analyzed with real-time PCR in HSCs.The expressions of SIRT1,EZH2,Collagen I,α-SMA,E-Cadherin and Vimentin in AML-12 cells were assayed by Western Blot.Immunofluorescence(×400)analysis of E-Cadherin and Vimentin protein expressions were tested in AML-12 cells.(5)AML-12 cells were divided into four groups: negative control(si-control)group,si-control+CS(10μM)group,si-SIRT1 group and si-SIRT1+CS(10μM)group.The protein expressions of SIRT1,EZH2,acetyl-EZH2 were determined by Western Blot.(6)AML-12 cells were divided into five groups: negative control(si-control)group,si-control+TGFβ1(4ng/ml)group,si-control+TGFβ1(4ng/ml)+Res(resveratrol,10μM)group,si-control+TGFβ1(4ng/ml)+CS(10μM)group and si-SIRT1+TGFβ1(4ng/ml)group.The protein expressions of SIRT1,EZH2,acetyl-EZH2 were determined by Western Blot.(7)HSCs and AML-12 cells were divided into five groups: negative control(pc DNA3.1)group,pc DNA3.1+TGFβ1(4ng/ml)group,pc DNA3.1+TGFβ1(4ng/ml)+Res(10μM)group,pc DNA-EZH2+TGFβ1(4ng/ml)+Res(10μM)group.Cells were transfected with pc DNA3.1 or pc DNA-EZH2 plasmids for 48 h and then pretreated with Res for 6 h before TGFβ1 treatment for 12 h,as indicated.Real-time PCR was used to analyze the levels of TIMP1,CTGF,Collagen I and α-SMA in HSCs.The expressions of E-Cadherin and Vimentin in AML-12 cells were analyzed with Western Blot.Results: The levels of serum ALT and AST were distinctly increased in the rats injected with CCl4,and there were marked hepatocellular necrosis,collagen fibril generation in the CCl4 group.Fortunately,CS treatment markedly reduced these changes.CS significantly inhibited CCl4-and TGFβ1-induced liver fibrosis and reduced both HSC activation and epithelial-mesenchymal transition(EMT).Interestingly,the protective effect of CS was positively associated with SIRT1 activation and accompanied by EZH2 inhibition: SIRT1 activation significantly reduced the level of EZH2 acetylation,which decreases its stability,and thereby,inhibits HSC activation,reverses EMT and attenuates liver fibrosis.Conclusion: SIRT1/EZH2 plays an important hepatoprotective role in liver fibrosis.Activation of SIRT1 by CS inhibits HSC activation and reverses EMT in the progression of liver fibrosis through a novel mechanism involving EZH2 deacetylation,which affects its stability.Thus,the SIRT1/EZH2 pathway may represent an attractive pharmacological target for the development of drugs to arrest the progression of liver fibrosis,and CS maybe a new potential candidate for anti-fibrotic clinical therapy. |
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